Classical Swine Fever Virus (CSFV)
Classical swine fever virus (CSFV) is the causing agent of a highly contagious disease of pigs and wild boars, but is totally innocuous for humans. CSFV, together with bovine viral diarrhea virus and border disease virus of ovines, belongs to the genus pestivirus within the family Flaviviridae. The other members of the Flaviviridae are the genus flavivirus (prototype: yellow fever virus) and the genus hepacivirus (hepatitis C virus).
Pestiviruses are small enveloped viruses with a single-stranded 12.3 kb RNA genome of positive polarity containing one large open reading frame (ORF) flanked by 5′ and 3′ nontranslated regions. The ORF encodes a polyprotein that is composed of four structural and eight nonstructural (NS) proteins in the order NH2—Npro -C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (FIG. 1A). The structural components of the virion include the capsid protein C and the three envelope glycoproteins Ems, E1, and E2. The leader autoprotease NPpro as well as Erns—a glycoprotein with ribonuclease activity, which is both secreted and virus-associated—are unique to the genus pestivirus. NPpro is considered an accessory protein not required for virus replication, but having a role in the pathogenesis of the virus due to its capacity to interfere with the innate immune response. Furthermore, Npro is an autoprotease which cleaves itself off the nascent viral polyprotein thereby generating the amino-terminus of the capsid protein C. Proteins NS3 to NS5B are sufficient for replication of the viral RNA and are considered to form the RNA replication complex. Uncleaved NS2-NS3 as well as p7 are required for virus particle formation.
Manipulation of the genome of RNA viruses is only possible if corresponding cDNA clones, preferably inserted in plasmids, are available for stable propagation of the genetic information in bacterial cells. These clones are designed to allow in vitro transcription of authentic infectious viral RNA that can be transfected into susceptible cells where the viral RNA will replicate and eventually give rise to the generation of infectious virus. Over the past 15 years cDNA clones have been established for many viruses of the family Flaviviridae, including pestiviruses.
Following on the development of the pestivirus cDNA clones, a recombinant CSFV derived from strain Alfort/187 (Ruggli et al., J Virol 70, 3478-3487, 1996) with a stably inserted chloramphenicol acetyl transferase (CAT) gene was generated (Moser et al., J Virol 72, 5318-5322, 1998), demonstrating that a foreign gene can be expressed in fusion with the NApro protein without affecting virus replication. Also, replacement of the entire Npro gene in CSFV by the murine ubiquitin gene did not impair the replication ability of the virus (Tratschin et al., J Virol 72, 7681-7684, 1998).
CSFV Replicons
By definition, a replicon represents a molecule that replicates autonomously in host cells. Therefore, the standard, full-length RNA genome of CSFV is the prototype CSFV replicon. Investigations using engineered cDNA of CSFV have been performed to determine the viral proteins required for RNA replication. This allowed the conclusion that the replication complex is composed of the 5 proteins NS3, NS4A, NS4B, NS5A, and NS5B. Subsequent to the above work, artificial replicons lacking the coding sequence for only one or several of the structural proteins, C, Erns, E1, or E2, were generated (Frey et al., Vet Res 37, 655-70, 2006). Such replicons have to be complemented in cell lines constitutively expressing the respective, missing structural protein(s) of the virus, allowing the rescue of viral particles carrying the defective (replicon) genome. These defective CSFV particles are referred to as virus replicon particles (VRP).
CSFV Replicons as a Vaccine
CSFV replicons, in the form of the VRP, have been employed as experimental vaccines against CSFV in pigs, but not as vectors for human or other mammalian vaccination. VRP are infectious virions that contain subgenomic RNA with specific deletion(s) in at least one of the genes encoding the viral structural proteins. Such RNA replicates and expresses the encoded viral proteins in the host cell. However, due to the defect in at least one of the envelope proteins it cannot generate progeny VRP. VRP carrying a genome devoid of either the Ems or the E2 gene have recently been proposed as live-attenuated, non-transmissible CSF vaccines. Challenge with highly virulent CSFV of pigs parenterally vaccinated with VRP have shown that VRP lacking the Erns gene can be protective (Frey et al., Vet Res 37, 655-70, 2006). It was also noted that these VRP vaccines have the potential to induce both humoral and cell-mediated immunity.
Many vaccines in use today are based on inactivated virus, which tends to favor humoral immunity with little or no cell-mediated defense development. This may allow for the removal of free virus and some virus-infected cells through the action of antibodies, but antibodies cannot interact with virus in all compartments of the body. It is the cell-mediated immunity that can ensure removal of small foci of virus infected cells. Accordingly, if a vaccine could induce both humoral and cell-mediated immunity, the likelihood of a more solid immune defense would be forthcoming.